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Cloning and Analysis of a 7α,15α Dihydroxylated dhea P450 Enzyme Gene

A DHEA, hydroxylation technology, applied in genetic engineering, oxidoreductase, DNA preparation, etc., can solve the problems of whole-cell catalysis prone to produce by-products, long conversion cycle, etc.

Active Publication Date: 2015-09-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

used in this patent C. lini ST-1 can generate a large amount of 7α,15α-diOH-DHEA when DHEA is used as a substrate, but its conversion cycle is long, and the whole cell catalysis is easy to produce by-products

Method used

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  • Cloning and Analysis of a 7α,15α Dihydroxylated dhea P450 Enzyme Gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Spirospora flaxensis ( Colletotrichum lini ) Extraction of ST-1 total RNA

[0033] C. liniST-1 was cultured in enzyme-producing medium at 30°C, 220 rpm for 2 days. Collect the bacteria by vacuum filtration, wash 2-3 times with sterile water, put the wet bacteria in a pre-cooled mortar, add 1 mL Trizol reagent, homogenize at low temperature for 2 min; transfer the homogenate to a 1.5 mL centrifuge tube Add 0.2 mL of chloroform, shake vigorously for 30 s, place at room temperature for 3 min, centrifuge at 12,000 rpm at 4 °C for 10 min; transfer the upper aqueous phase to a clean centrifuge tube, add 1 / 2 times absolute ethanol (v / v) , mix well; use a pipette to transfer the mixture to the adsorption column, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 3 minutes, discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, add 500 μL RPE solution, let it stand for 2 minutes, centri...

Embodiment 2

[0034] Embodiment 2 Spirospora flaxensis ( Colletotrichum lini ) Cloning of ST-1 3' mRNA sequence

[0035] by C. lini ST-1 total RNA was used as a template, and AMV reverse transcriptase was used to perform reverse transcription reaction to synthesize the first strand of cDNA. The reverse transcription reaction was carried out according to the following conditions: incubation at 42-60°C for 15-30 minutes, inactivation of AMV reverse transcriptase at 99°C for 5 minutes, and storage at 5°C for 5 minutes.

[0036] In the reverse transcription tube add Ex Taq For HS enzyme, perform the first round of PCR with M13 Primer M4 and FP1 as primers at the same time. The reaction conditions are 94°C for 2 min, 30 cycles (94°C for 30s, 52°C for 30s, 72°C for 45s), and 72°C for 10min. The PCR product was analyzed by 2% agarose gel electrophoresis, the target band was recovered and ligated with PMD19-T (PMD19-T-CYPX), transformed into JM109, and sent to Shanghai Sangon for sequencing a...

Embodiment 3

[0037] Embodiment 3 Spirospora flaxensis ( Colletotrichum lini ) Cloning of ST-1 5' end mRNA sequence

[0038] The first round of amplification was performed with the 5'-end RACE Outer Primer and RP1 as primers, and the reaction conditions were: 94°C for 3 min, 30 cycles (94°C for 30 s, 65°C for 30 s, 72°C for 90 s), 72°C for 10 min ; Use Inner Primer and RP2 as primers for the second round of PCR. The reaction conditions are: 94°C for 3 min, 30 cycles (94°C for 30s, 55°C for 30s, 72°C for 90s, and 72°C for 10 min. The two rounds of PCR products Analyzed by 1% agarose gel electrophoresis, the target band was recovered by tapping the gel and ligated with PMD19-T (PMD19-T-CYPX5'), transformed into JM109, and then sent to Shanghai Synergy for sequencing.

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Abstract

The invention provides a cloning and analysis method for complete mRNA and DNA sequences of a novel fungal cytochrome P450 enzyme CYP X from Colletotrichum lini ST-1. The nucleotide sequences are SEQ ID NO:1 and SEQ ID NO:2 respectively, and a corresponding amino acid sequence is named as SEQ ID NO:3. The enzyme can efficiently convert DHEA into 7alpha, 15alpha-diOH-DHEA. Obtaining of the gene lays the theoretical foundation for heterologous expression and industrial production. The gene has great application potential and economic value, and also lays the theoretical foundation for related research on cytochrome P450 enzyme.

Description

technical field [0001] The design of the present invention is derived from the spirochete sp. flaxensis ( Colletotrichum lini ) A P450 enzyme capable of 7α, 15α-dihydroxylation of DHEA in the ST-1 strain, including the cloning and analysis of its complete mRNA and DNA sequences, belongs to the technical field of bioengineering. Background technique [0002] Since the application of biotransformation technology in the study of steroid hydroxylation, a large number of hydroxysteroid compounds with important pharmacological effects and medicinal value have been discovered, and steroid hydroxylation is still one of the hot spots in the study of steroid drugs. Some 3-hydroxysteroids, such as dehydroepiandrosterone (DHEA), cholesterol, and pregnenol, are mainly converted into corresponding hydroxysteroid derivatives in the organs and tissues of the human body, and have important biological activity. Among them, the dihydroxylation product of dehydroepiandrosterone (DHEA), 7α, 1...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/10C12R1/645
CPCC12N9/0081C12N15/1031C12Y114/15006
Inventor 许正宏史劲松李会李恒张旦旦许鸿瑜其他发明人请求不公开姓名
Owner JIANGNAN UNIV
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